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Research Article
Article History
Received : 08.04.2015
Revised : 15.05.2015
Accepted : 20.05.2015
1. Introduction
K. Saranya/ Life Science Archives (LSA), Volume 1, Issue 3, Page 186 - 191, 2015
antibiotics (around 80% of the total antibiotic
production) and active secondary metabolites.
Actinomycetes, the filamentous bacteria, are
primarily saprophytic microorganisms of the soil,
where they contribute significantly to the turnover
of complex biopolymers, such as lignocellulose,
hemicellulose, pectin, etc. (Vijayakumar et al.,
2007).
The microbial fibrinolytic enzymes,
especially those from actinomycetes, have the
potential to be developed as drugs to prevent
cardiovascular diseases (Sasirekha et al., 2012).
Early evidence supporting the existence of marine
actinomycetes came from the description of
Rhodococcus marinonascene the first marine
actinomycetes species to be characterized
(Helmke and Weyland, 1984). They produce
numerous substances essential for health such as
antibiotics, enzymes, immunomodulators, etc. The
productivity of Streptomyces strains as antibiotic
producers remains unique amongst Actinomycetes
strains. Early evidence supporting the existence of
marine actinomycetes came from the description
of Rhodococcus marinonascene, the first marine
actinomycetes species to be characterized. The
aim of this work was to isolate and characterize
specific antibiotic producing actinomycetes strain
from marine soil sample.
2. Materials and Methods
Sample collection
The samples were collected from the coastal
region of Bay of Bengal near the shore regions of
Kanathur of Chennai, India. . The samples were
kept for drying in room temperature
Isolation, Characterization and identification
Actinomycetes
187
and
preservation
of
K. Saranya/ Life Science Archives (LSA), Volume 1, Issue 3, Page 186 - 191, 2015
medium was cooled and the loop full of individual
strains was taken and inoculated separately into
the Yeast Malt Broth. That broth was kept in an
orbital shaker for incubation for 10 days to obtain
for maximum growth of actinomycetes. The
culture was filtered and centrifuged at 10,000 rpm
for 10 min. The supernatant was collected and
addition of equal volume of ethyl acetate was
added. The mixture was kept in separating funnel
and shaken for 1 hour. After 24 hours the upper
layer was collected and kept in condensation at 40
C in round bottom flask. The extract was obtained
and it was carefully poured on to sterile petriplates
allowed for drying for 2 days. The plates were
scrapped using 1 ml of ethyl acetate and the
extract was transferred to sterile tubes.
Partial purification of secondary metabolites
The concentrated ethyl acetate extract was
subjected to TLC analysis using mobile phase
ethyl acetate: hexane in the ratio. The crude
extract was spotted on pre-coated silica sheet (5
cm 1.5 cm) using capillary tube. After
separation of compounds, the TLC sheet was air
dried and visualized under UV and iodine.
Screening
of
Actinomycetes
antioxidant
activity
of
100
188
K. Saranya/ Life Science Archives (LSA), Volume 1, Issue 3, Page 186 - 191, 2015
control. The intensity of the color formed was
measured spectroscopically at 412 nm against
reagent blank. The % hydroxyl radical scavenging
activity was calculated by the following formula:
% HRSA = from [(A0 A1)/A0] 100, where A0
is the absorbance of the control and A1 is the
absorbance of the extract/standard.
Cytotoxicity Assay on cancer cell lines
Chemicals and reagents
MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) in vitrogen, USA.
Acridine orange were obtained from Sigma, USA.
All other fine chemicals were obtained from
SigmaAldrich, St. Louis.
Cell culture
MCF 7 cells obtained from NCCS
(National Centre For Cell Science, Pune) were
cultured in Rose well Park Memorial Institute
medium (RPMI), supplemented with 10% fetal
bovine serum, penicillin/streptomycin (250 U/ml),
gentamycin (100 g/ml) and amphotericin B (1
mg/ml) were obtained from Sigma Chemicals,
MO, USA. All cell cultures were maintained at
37C in a humidified atmosphere of 5% CO2.
Cells were allowed to grow to confluence over 24
hrs before use.
Cell growth inhibition studies by MTT assay
Cell viability was measured with the
conventional MTT reduction assay, as described
previously with slight modification. Briefly, MCF
7 cells were seeded at a density of 5 103
cells/well in 96 - well plates for 24 hrs, in 200 l
of RPMI with 10% FBS. Then, culture supernatant
was removed and RPMI containing various
concentrations (1 100 g/mL) of test compound
was added and incubated for 48 hrs. After
treatment cells were incubated with MTT (10 l, 5
mg/ml) at 37 C for 4 hrs and then with DMSO at
room temperature for 1 hr. The plates were read at
595
nm
on
a
scanning
multi-well
spectrophotometer. Data represented the mean
189
K. Saranya/ Life Science Archives (LSA), Volume 1, Issue 3, Page 186 - 191, 2015
Phospho molybdenum assay
The ethyl acetate extract of strain - 8 was
used to determine its antioxidant capacities by the
formation of light green phosphomolybdenum
complex. We can observe from the table that
sample has moderate phosphomolybdenum
reducing potential with an increase in the OD to
value from 0.117 to 0.171.
Table 1: Phosphomolybdenum reducing potential
S.
No
1
2
3
4
5
6
Concentration
(g/ml)
20
40
60
80
100
Control
OD
190
Production
of
antioxidant
metabolites from strain-8
secondary
0.117
0.134
0.149
0.156
0.171
0.093
Concentration
(g/ml)
20
40
60
80
% MCA
24.83
29.11
51.69
77.42
HRSA assay
The potential of the compounds to
scavenge hydroxyl radical was observed to be in
the range of 38 57 %.
Table 3: Hydroxyl radical Scavenging potential
S.
No
1
2
3
4
5
Concentration
(g/ml)
20
40
60
80
100
%HRSA
38.31
39.25
48.13
53.27
57.94
S.
No
1
2
3
4
5
Concentration
(g/ml)
1
10
20
50
100
Cell viability
(%)
96.66
95.37
84.34
64.26
52.24
K. Saranya/ Life Science Archives (LSA), Volume 1, Issue 3, Page 186 - 191, 2015
metabolite showed 83 % inhibition at 0.2 mg/ml,
and the inhibition was compared with the standard
antioxidant ascorbic acid which showed 84%
inhibition at 0.2mg/ ml concentration. The
potential strain was identified as Streptomyces
species and designated as Streptomyces sp.
However, the secondary metabolite showed 81%
inhibition at 10 mg/ml.
4. Conclusion
Because of the immense biological
diversity in the sea as a whole, it is increasingly
recognized that a large number of novel chemical
entities exists in the oceans. As marine
microorganisms, particularly actinomycetes, have
evolved the greatest genomic and metabolic
diversity, efforts should be directed towards
exploring marine actinomycetes as a source for the
discovery of novel secondary metabolites. In this
respect, future success relies on our ability to
isolate novel actinomycetes from the marine
environments. Recent investigations using
enrichment techniques, new selection methods and
media have led to the isolation of novel
actinomycetes from sediment samples. Improved
recovery yields of marine actinomycetes from
sponges using nutrient supplements and enzymes
have been reported. These recent successes are the
first step in the right direction. Further
development work in improving isolation
strategies in the recovery of marine actinomycetes
is of utmost importance for ensuring success in
this area.
5. References
1) Attimarad,
Asif,
Kalyani
and
Nageshchandrashekhar, 2012. Screening,
isolation and purification of antibacterial
agents
from
marine
actinomycetes.
International
Current
Pharmaceutical
Journal, 1(12): 394 - 402.
2) Bull AT, Stach JEM, Ward AC, Goodfellow
M. 2005. Marine actinobacteria: perspectives,
challenges, future directions. Antonie Van
Leeuwenhoek, 87: 65 - 79.
3) Devi NKA, Jeyarani M, Balakrishnan. K.
2006. Isolation and identification of marine
actinomycetes and their potential in
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